Time serial transcriptome reveals Cyp2c29 as a key gene in hepatocellular carcinoma development.
ABSTRACT: Objective: Hepatocellular carcinoma (HCC) is a severely lethal cancer that usually originates from chronic liver injury and inflammation. Although progress on diagnosis and treatment is obvious, the cause of HCC remains unclear. In this study, we sought to determine key genes in HCC development. Methods: To identify key regulators during HCC progression, we performed transcriptome sequencing to obtain time series gene expression data from a mouse model with diethylnitrosamine-induced liver tumors and further verified gene expression and function in vitro and in vivo. Results: Among the differentially expressed genes, Cyp2c29 was continuously downregulated during HCC progression. Overexpression of Cyp2c29 suppressed NF-?B activation and proinflammatory cytokine production by increasing the production of 14,15-epoxyeicosatrienoic acid in vitro. Furthermore, overexpression of Cyp2c29 in vivo protected against liver inflammation in mouse models of liver injury induced by both acetaminophen and CCl4. Two human homologs of mouse Cyp2c29, CYP2C8 and CYP2C9, were found to be downregulated in human HCC progression, and their expression was positively correlated with overall survival in patients with HCC (significance: P = 0.046 and 0.0097, respectively). Conclusions: Collectively, through systematic analysis and verification, we determined that Cyp2c29 is a novel gene involved in liver injury and inflammation, which may be a potential biomarker for HCC prevention and prognosis determination.
Project description:Cell death is a key driver of disease progression and carcinogenesis in chronic liver disease (CLD), highlighted by the well-established clinical correlation between hepatocellular death and risk for the development of cirrhosis and hepatocellular carcinoma (HCC). Moreover, hepatocellular death is sufficient to trigger fibrosis and HCC in mice. However, the pathways through which cell death drives CLD progression remain elusive. Here, we tested the hypothesis that high-mobility group box 1 (HMGB1), a damage-associated molecular pattern (DAMP) with key roles in acute liver injury, may link cell death to injury responses and hepatocarcinogenesis in CLD. While liver-specific HMGB1 deficiency did not significantly affect chronic injury responses such as fibrosis, regeneration, and inflammation, it inhibited ductular/progenitor cell expansion and hepatocyte metaplasia. HMGB1 promoted ductular expansion independently of active secretion in a nonautonomous fashion, consistent with its role as a DAMP. Liver-specific HMGB1 deficiency reduced HCC development in 3 mouse models of chronic injury but not in a model lacking chronic liver injury. As with CLD, HMGB1 ablation reduced the expression of progenitor and oncofetal markers, a key determinant of HCC aggressiveness, in tumors. In summary, HMGB1 links hepatocyte death to ductular reaction, progenitor signature, and hepatocarcinogenesis in CLD.
Project description:Hepatic stellate cells (HSCs) play critical roles in liver fibrosis and hepatocellular carcinoma (HCC). Vitamin D receptor (VDR) activation in HSCs inhibits liver inflammation and fibrosis. We found that p62/SQSTM1, a protein upregulated in liver parenchymal cells but downregulated in HCC-associated HSCs, negatively controls HSC activation. Total body or HSC-specific p62 ablation potentiates HSCs and enhances inflammation, fibrosis, and HCC progression. p62 directly interacts with VDR and RXR promoting their heterodimerization, which is critical for VDR:RXR target gene recruitment. Loss of p62 in HSCs impairs the repression of fibrosis and inflammation by VDR agonists. This demonstrates that p62 is a negative regulator of liver inflammation and fibrosis through its ability to promote VDR signaling in HSCs, whose activation supports HCC.
Project description:The increase of the sedentary lifestyle and high-calorie diet have modified the etiological landscape of hepatocellular carcinoma (HCC), with a recrudescence of non-alcoholic fatty liver disease (NAFLD), especially in Western countries. The purpose of our study was to evaluate the impact of high-fat diet feeding on non-alcoholic steatohepatitis (NASH) establishment and HCC development. Streptozotocin-induced diabetic male mice were fed with high-fat-high-cholesterol diet (HFHCD) or high-fat-high-sugar diet (HFHSD) from 1 to 16 weeks. Even if liver tumors appear regardless of the high-fat diet, two distinct physiopathological patterns were evidenced, with much more severe NASH hallmarks (liver injury, inflammation and fibrosis) in diabetic mice fed with HFHCD. The mild hepatic injury, weak inflammation and fibrosis observed in HFHSD were interestingly associated with earlier emergence of more numerous liver tumors. When activated helper and cytotoxic T cells, detected by flow cytometry, infiltrated the liver of HFHCD-fed diabetic mice, a delay in the appearance of tumor nodules and a limitation of their numbers were observed, suggesting that the immune activities partly controlled tumor emergence. These data highlighted two different mouse models of HCC progression in diabetic mice depending on diet, which could be useful to evaluate new therapeutic approaches for HCC by targeting the immune response.
Project description:Hepatocyte death is associated with liver inflammation, fibrosis and hepatocellular carcinoma (HCC). Damaged cells trigger inflammation through activation of Toll-like receptors (TLRs). Although the role of TLR4 in HCC development has been reported, the role of TLR9 in the development of HCC remains elusive. To investigate the role of TLR4 and TLR9 signaling in liver inflammation-fibrosis-cancer axis, we took advantage of mice with hepatic deletion of transforming growth factor-?-activated kinase 1 (Tak1?Hep) that develop spontaneous liver injury, inflammation, fibrosis, and HCC, recapitulating the pathology of human HCC. We generated double knockout mice lacking genes of our interest with hepatic Tak1. Tak1?Hep mice and Tlr4-deficient Tak1?Hep mice had similar serum ALT levels, but Tlr4-deficient Tak1?Hep mice exhibited significantly reduced macrophage infiltration, myofibroblast activation and tumor formation. Ablation of TLR9 reduced spontaneous liver injury, inflammation, fibrosis, and cancer development in Tak1?Hep mice. In addition, the common adaptor, myeloid differentiation factor 88 (MyD88)-deficient Tak1?Hep mice also attenuated liver injury, macrophage recruitment, collagen deposition, and tumor growth compared with control Tak1?Hep mice. Genetic ablation of TNF receptor type I (TNFR) in Tak1?Hep mice remarkably reduced liver inflammation-fibrosis-cancer axis. Surprisingly, disruption of interleukin-1 receptor (IL-1R) had no effect on liver injury and tumor formation, although Il1r-deficient Tak1?Hep showed attenuated macrophage infiltration and collagen deposition. In conclusion, TLR4- and TLR9-MyD88 are driving forces of progression to HCC accompanied by liver inflammation and fibrosis in Tak1?Hep mice. Importantly, TLR4 and TLR9 downstream TNFR, but not IL-1R signaling is crucial for the development of HCC in Tak1?Hep mice.
Project description:Hepatocellular carcinoma (HCC) has become a leading cause of cancer-related death, making the elucidation of its underlying mechanisms an urgent priority. Inflammation is an adaptive response to infection and tissue injury under strict regulations. When the host regulatory machine runs out of control, nonresolving inflammation occurs. Nonresolving inflammation is a recognized hallmark of cancer that substantially contributes to the development and progression of HCC. The HCC-associated inflammation can be initiated and propagated by extrinsic pathways through activation of pattern-recognition receptors (PRRs) by pathogen-associated molecule patterns (PAMPs) derived from gut microflora or damage-associated molecule patterns (DAMPs) released from dying liver cells. The inflammation can also be orchestrated by the tumor itself through secreting factors that recruit inflammatory cells to the tumor favoring the buildup of a microenvironment. Accumulating datas from human and mouse models showed that inflammation promotes HCC development by promoting proliferative and survival signaling, inducing angiogenesis, evading immune surveillance, supporting cancer stem cells, activating invasion and metastasis as well as inducing genomic instability. Targeting inflammation may represent a promising avenue for the HCC treatment. Some inhibitors targeting inflammatory pathways have been developed and under different stages of clinical trials, and one (sorafenib) have been approved by FDA. However, as most of the data were obtained from animal models, and there is a big difference between human HCC and mouse HCC models, it is challenging on successful translation from bench to bedside.
Project description:Nonalcoholic fatty liver disease is the most common chronic liver disease and may progress to nonalcoholic steatohepatitis (NASH) and hepatocellular carcinoma (HCC). The molecular determinants of this pathogenic progression, however, remain largely undefined. Since liver tumorigenesis is driven by apoptosis, we examined the effect of overt hepatocyte apoptosis in a mouse model of NASH using mice lacking myeloid cell leukemia 1 (Mcl1), a pro-survival member of the BCL-2 protein family. Hepatocyte-specific Mcl1 knockout (Mcl1?hep) mice and control littermates were fed chow or FFC (high saturated fat, fructose, and cholesterol) diet, which induces NASH, for 4 and 10 months. Thereafter, liver injury, inflammation, fibrosis, and tumor development were evaluated biochemically and histologically. Mcl1?hep mice fed with the FFC diet for 4 months displayed a marked increase in liver injury, hepatocyte apoptosis, hepatocyte proliferation, macrophage-associated liver inflammation, and pericellular fibrosis in contrast to chow-fed Mcl1?hep and FFC diet-fed Mcl1-expressing littermates. After 10 months of feeding, 78% of FFC diet-fed Mcl1?hep mice developed liver tumors compared to 38% of chow-fed mice of the same genotype. Tumors in FFC diet-fed Mcl1?hep mice were characterized by cytologic atypia, altered liver architecture, immunopositivity for glutamine synthetase, and histologically qualified as HCC. In conclusion, this study provides evidence that excessive hepatocyte apoptosis exacerbates the NASH phenotype with enhancement of tumorigenesis in mice.
Project description:Glycine-N-methyltransferase (GNMT) is essential to preserve liver homeostasis. Cirrhotic patients show low expression of GNMT that is absent in hepatocellular carcinoma (HCC) samples. Accordingly, GNMT deficiency in mice leads to steatohepatitis, fibrosis, cirrhosis, and HCC. Lack of GNMT triggers NK cell activation in GNMT(-/-) mice and depletion of TRAIL significantly attenuates acute liver injury and inflammation in these animals. Chronic inflammation leads to fibrogenesis, further contributing to the progression of chronic liver injury regardless of the etiology. The aim of our study is to elucidate the implication of TRAIL-producing NK cells in the progression of chronic liver injury and fibrogenesis. For this we generated double TRAIL(-/-)/GNMT(-/-) mice in which we found that TRAIL deficiency efficiently protected the liver against chronic liver injury and fibrogenesis in the context of GNMT deficiency. Next, to better delineate the implication of TRAIL-producing NK cells during fibrogenesis we performed bile duct ligation (BDL) to GNMT(-/-) and TRAIL(-/-)/GNMT(-/-) mice. In GNMT(-/-) mice, exacerbated fibrogenic response after BDL concurred with NK1.1(+) cell activation. Importantly, specific inhibition of TRAIL-producing NK cells efficiently protected GNMT(-/-) mice from BDL-induced liver injury and fibrogenesis. Finally, TRAIL(-/-)/GNMT(-/-) mice showed significantly less fibrosis after BDL than GNMT(-/-) mice further underlining the relevance of the TRAIL/DR5 axis in mediating liver injury and fibrogenesis in GNMT(-/-) mice. Finally, in vivo silencing of DR5 efficiently protected GNMT(-/-) mice from BDL-liver injury and fibrogenesis, overall underscoring the key role of the TRAIL/DR5 axis in promoting fibrogenesis in the context of absence of GNMT. Overall, our work demonstrates that TRAIL-producing NK cells actively contribute to liver injury and further fibrogenesis in the pathological context of GNMT deficiency, a molecular scenario characteristic of chronic human liver disease.
Project description:Aldo-keto reductase 1B10 (AKR1B10), a member of aldo-keto reductase superfamily, contributes to detoxification of xenobiotics and metabolization of physiological substrates. Although increased expression of AKR1B10 was found in hepatocellular carcinoma (HCC), the role of AKR1B10 in the development of HCC remains unclear. This study aims to illustrate the role of AKR1B10 in hepatocarcinogenesis based on its intrinsic oxidoreduction abilities. HCC cell lines with AKR1B10 overexpression or knockdown were treated with doxorubicin or hydrogen peroxide to determinate the influence of aberrant AKR1B10 expression on cells' response to oxidative stress. Using Akr1b8 (the ortholog of human AKR1B10) knockout mice, diethylnitrosamine (DEN) induced liver injury, chronic inflammation and hepatocarcinogenesis were explored. Clinically, the pattern of serum AKR1B10 relevant to disease progression was investigated in a patient cohort with chronic hepatitis B (n=30), liver cirrhosis (n=30) and HCC (n=40). AKR1B10 expression in HCC tissues was analyzed using both the TCGA database (n=371) and our collected HCC samples (n=67). AKR1B10 overexpression reduced hepatocyte injury while AKR1B10 knockdown augmented reactive oxygen species (ROS) accumulation and apoptotic cell death. Consistently, Akr1b8 deficiency in mice promoted DEN-induced hepatocyte damage and liver inflammation characterized by increased phospho-H2AX, serum alanine aminotransferase, interleukin-6 and tumor necrosis factor alpha level, myeloid cell infiltration and led to more severe hepatocarcinogenesis and metastasis compared with wild type mice due to significant alteration on detoxification and oxidoreduction. AKR1B10 was compensatory expressed and gradually upregulated in the process of liver disease progression in HCC and increased oxidative stress upregulated AKR1B10 through NRF2. Our results here suggested that through oxidoreduction and detoxification, AKR1B10 played an important role in protecting hepatocytes from damage induced by ROS. Deficiency of AKR1B10 might accelerate hepatotoxin and inflammation-associated hepatocarcinogenesis. AKR1B10 expression elevation in HCC could be a result of compensatory upregulation, rather than a driver of malignant transformation during the development of HCC.
Project description:Chronic infection of Hepatitis B Virus (HBV) is one of the highest risk factors of hepatocellular carcinoma (HCC). The accumulation of HBV surface antigen (HBsAg) into hepatocytes induces inflammation and oxidative stress, impairing their replicative ability and allowing the activation of the hepatic stem cells (SC) compartment. This study aimed to understand the involvement of SC during hepatocarcinogenesis in HBsAg-related liver damage, from early injury until HCC. HBsAg-transgenic (TG) and wild type (WT) mouse were followed at several stages of the liver damage: inflammation, early hepatocytes damage, dysplasia, and HCC. Serum transaminases, liver histology, and diagnostic data were collected. The expressions of SC and cancer stem cells (CSC) markers was analyzed by RT-qPCR, immunohistochemistry and Western blot. Starting from 3 months, TG animals showed a progressive liver damage characterized by transaminases increase. The up-regulations of SCs markers Cd34 and Sca-1 started from the beginning of the inflammatory stage while progressive increase of Krt19 and Sox9 and CSCs markers Epcam and Cd133 from early hepatic injury. The expressions of Cd133, Cd34, and Afp were significantly higher in HCC compared to paired non-HCC tissue, in contrast to Epcam and Krt19. Western blot and IHC confirmed the positivity of Cd34 and Cd133 in small cells subpopulation.
Project description:Chronic liver injury triggers liver fibrosis and hepatocellular carcinoma (HCC), the third leading cause of cancer-related mortality. Cyclin E1 (CcnE1, formerly designated Cyclin E) is a regulatory subunit of the Cyclin-dependent kinase 2 (CDK2). It is overexpressed in approximately 70% of human HCCs correlating with poor prognosis, while the relevance of its orthologue Cyclin E2 (CcnE2) is unclear. Hepatocyte-specific deletion of NF-kappa-B essential modulator (NEMOΔhepa) leads to chronic hepatitis, liver fibrosis, and HCC as well as CcnE upregulation. To this end, we generated NEMOΔhepa/CcnE1-/- and NEMOΔhepa/CcnE2-/- double knockout mice and investigated age-dependent liver disease progression in these animals. Deletion of CcnE1 in NEMOΔhepa mice decreased basal liver damage and reduced spontaneous liver inflammation in young mice. In contrast, loss of CcnE2 did not affect liver injury in NEMOΔhepa livers pointing to a unique, non-redundant function of CcnE1 in chronic hepatitis. Accordingly, basal compensatory hepatocyte proliferation in NEMOΔhepa mice was reduced by concomitant ablation of CcnE1, but not after loss of CcnE2. In aged NEMOΔhepa mice, loss of CcnE1 resulted in significant reduction of liver tumorigenesis, while deletion of CcnE2 had no effect on HCC formation. CcnE1, but not its orthologue CcnE2, substantially contributes to hepatic inflammatory response, liver disease progression, and hepatocarcinogenesis in NEMOΔhepa mice.